Naked ZFNs Point to More Efficient, Safer Gene Therapy

Researchers have made the surprising discovery that DNA-cutting zinc finger nucleases (ZFNs) can directly penetrate into cells and don't need to be delivered as DNA constructs using vectors. The discovery, by Carlos F Barbas III, Ph.D., and colleagues at The Scripps Research Institute, could feasibly lead to the development of much simpler and safer ways of editing the genetic content of either stem cells or differentiated cells developed as treatments for diseases including HIV.

Reporting in Nature Methods, the Scripps investigators say they have found that the direct delivery of ZFNs as proteins to a range of mammalian cell types leads to efficient endogenous gene disruption with minimal off-target effects. The approach was successful in primary adult human fibroblasts that are commonly used to generate induced pluripotent stem cells, as well as hard to transfect cells such as primary CD4+ T cells and patient-derived leukemia cell lines. The work is described in a paper titled “Targeted gene knockout by direct delivery of zinc-finger nuclease proteins.”

ZFNs are essentially DNA scissors that generate double-stranded breaks (DSBs) at a designated DNA sequence. Used to effect precise genetic modifications, the synthetic fusion proteins consist of an engineered zinc finger DNA-binding domain (zinc finger protein, or ZFP) fused to the cleavage domain of the FokI restriction endonuclease. The Barbas laboratory at Scripps developed the original technology for constructing the ZFPs used to direct the nuclease to its DNA target site.

Getting ZFNs into cells currently involves transfection with viral or nonviral vectors carrying the ZFN gene. However, this approach isn’t ideal, both because of the time and expense in generating the gene construct and vector, and also because the vector-based approach can result in undesirable side effects such as insertional mutagenesis or toxicity.

In their search for an improved method for delivering ZFNs into cells, the Barbas team found that the ZFN proteins themselves can directly penetrate into cells and effect genetic modification without the unwanted side effects associated with vector-delivered ZFN genes. “We tried working with unmodified ZFNs, and lo and behold, they were easy to produce and entered cells quite efficiently,” Barbas states.

In their published paper the team reports that ZFNs can make their own way into a range of human cell types, including those that resist vector-based delivery. One set of tests demonstrated that a ZFN protein targeting the CCR5 gene (which is required by HIV to infect immune system cells) was efficiently taken up into human T cells and led to rapid disruption of CCR5 gene activity. This has particular clinical relevance because clinical trials are currently under way to evaluate a gene therapy that uses ZFNs to disrupt CCR5 genes in T cells that are infused back into the patient.

Notably, the Barbas team found that in comparison with the ZFN gene-based approach to CCR5 disruption, the delivery of ZFN proteins to T cells appeared much safer. “At some off-target locations where the gene therapy approach frequently causes damage, we saw no damage at all from this new technique,” Dr Barbas states. The team has a particular interest in HIV therapy, and is looking to develop an anti-CCR5 hematopoietic stem cell therapy for HIV. “Even a small number of stem cells that carry this HIV-resistance feature could end up completely replacing a patient’s original and vulnerable T cell population,” Barbas adds.

Direct delivery of ZFNs could also feasibly allow the direct engineering of hematopoietic stem cells or cells differentiated from fibroblast-derived induced pluripotent stem cells as the basis for autologous therapies for a range of diseases. And potential applications of the technique for effecting precise genome editing for disease research are manifold.

“In contrast to methods that require ZFN expression from DNA, ZFN protein delivery leads to comparatively fewer off-target cleavage events and does not carry the risk of insertional mutagenesis,” the authors conclude. “Thus, this method is suitable for genome-editing applications in which minimizing cellular toxicity or maintaining genetic integrity is of particular importance, such as the in vitromodeling of human diseases and the ex vivomodification of nontransformed human cell types.”